LDH-stabilized ultrasmall iron oxide nanoparticles as a platform for hyaluronidase-promoted MR imaging and chemotherapy of tumors

Development of unique theranostic nanoplatforms for tumor imaging and therapy remains an active topic in current nanomedicine. Here, we designed a novel targeted theranostic nanoplatform for enhanced T1-weighted magnetic resonance (MR) imaging-guided chemotherapy by constructing layered double hydroxide (LDH)-stabilized ultrasmall iron oxide (Fe3O4) nanoparticles with hyaluronic acid (HA) modified as targeting agents, and anticancer drug doxorubicin (DOX) loaded with a high loading efficiency. Methods: The structure and release property of LDH-Fe3O4-HA/DOX nanoplatforms were characterized systematically. B16 melanoma cells with CD44 receptors overexpressed were used as model cells to determine the biocompatibility, targeting capability, and therapeutic efficiency of nanoplatforms. For in vivo experiment, hyaluronidase (HAase) pretreatment was combined with nanoplatform administration to investigate the MR imaging and chemotherapeutic effect. Results: The LDH-Fe3O4-HA nanohybrids possess good colloidal stability and cytocompatibility, display an r1 relaxivity 10-fold higher than the pristine ultrasmall Fe3O4 (4.38 mM-1 s-1 vs 0.42 mM-1 s-1), and could release drug in a pH-responsive manner. In vitro experiments demonstrate that LDH-Fe3O4-HA/DOX nanohybrids are able to specifically target B16 cells overexpressing CD44 receptors and effectively release DOX to nucleus. In vivo results show that with the pretreatment of tumor tissue by HAase to degrade the overexpressed HA in extra-cellular matrix, the designed nanoplatforms have a better tumor penetration for significantly enhanced MR imaging of tumors and tumor chemotherapy with low side effects. Conclusion: The designed LDH-Fe3O4-HA/DOX nanohybrids may be developed as a novel targeted theranostic nanoplatform for enhanced T1-weighted MR imaging-guided chemotherapy of CD44 receptor-overexpressing tumors.

IR spectrophotometer (Thermo Electron Corporation, Madison, WI). Samples were mixed with milled KBr crystals and pressed to form 13-mm diameter disks before measurements. A TG 209 F1 (NETZSCH Instruments Co., Ltd., Bavaria, Germany) thermogravimetric analyzer was used for thermogravimetric analysis (TGA) of the samples under nitrogen atmosphere in a temperature range of 10-900°C. Zeta potential and dynamic light scattering (DLS) measurements were conducted using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, UK) equipped with a standard 633 nm laser. UV-vis spectrophotometer was carried out using a Lambda 25 UV-vis spectrophotometer (Perkin Elmer, Boston, MA). Mg, Al and Fe concentrations of these NPs in solutions were analyzed using a Leeman Prodigy inductively coupled plasma-optical emission spectroscopy (ICP-OES, Hudson, NH). T1 and T2 relaxometry were performed using a 0.5 T NMI20 Analyzing and Imaging system (Shanghai NIUMAG Corporation, Shanghai, China). The parameters were set as follows: TR = 400 ms, TE = 20 ms, resolution = 156 mm × 156 mm, section thickness = 0.5 mm. The r relaxivity was obtained through linear fitting of the inverse T relaxation time (1/T) as a function of Fe concentration.

Drug loading
The DOX encapsulation efficiency and percentage can be calculated by measuring the concentration of free DOX in the collected supernatants after 3 times of centrifugation using a Lambda 25 UV-vis spectrophotometer (PerkinElmer, Waltham, MA) at 480 nm and a standard DOX absorbance-concentration calibration curve. The drug loading content (DL%) and entrapment efficiency (EE%) of nanodisks can be calculated using eqn (1) and (2), respectively. (1) where Mt, M0 and ML stand for the masses of the encapsulated DOX, the initial DOX, and the drug loaded nanocomplexes, respectively.

In vitro drug release
In vitro drug release profile of DOX from LDH-Fe3O4-HA/DOX was investigated in the presence or absence of HAase at pH 5.0 and 7.4 buffer solutions. LDH-Fe3O4-HA/DOX were dispersed in 1 mL corresponding buffer solution at the concentration of 1 mg/mL with/without HAase (1 mg/mL), and then sealed in a dialysis bag (cutoff Mw = 3500). The dialysis bag was immersed in 9 mL buffer solution (pH 5.0 or 7.4) and placed in shaker at 37°C for 48 h. Within a specified time interval (0.5, 1, 2, 4, 6, 9, 12, 24, 48 h), 1 mL sample solution was taken out from buffer solution to measure the released amount of DOX (λDOX = 480 nm) by ultraviolet-visible spectrophotometer, and an equivalent amount of fresh buffer solution was added.
LDH-Fe3O4-HA were dispersed in buffer solution of pH 5.0 and 7.4 (1 mg/mL), and then HAase (1 mg/mL) was added to degrade HA on the surface of NPs at 37°C under shaking. The zeta potential of LDH-Fe3O4-HA were measured by DLS at 15 min, 1 h, 2 h, 4 h, respectively.

T1 and T2 MR relaxometry and T1 imaging measurement.
An NMI20-Analyst NMR Analyzing and Imaging system (0.5 T, Shanghai NIUMAG Corporation, Shanghai, China) was utilized to measure the T1 and T2 MR relaxometry of the Fe3O4, LDH-Fe3O4 and LDH-Fe3O4-HA NPs. First, these nanoparticles were formulated into the same iron concentration (0.1-1.6 mM), then the transverse relaxivity (r1 and r2) was calculated by linear fitting the inverse T1 and T2 relaxation time (1/T1 and 1/T2) as a function of Fe concentration, respectively.
Finally, according to their relaxation rate, comparing the introduction of LDH and HA has a positive effect on the relaxation rate of contrast agent. Consistent with the above results, concentration dependent brightening effects were also observed by T1-weighted MR images under the same gradient concentration conditions. After the mice were sacrificed at the given time points (15 min, 60 min and 24 h, respectively), the heart, liver, spleen, lung, kidney and tumor were extracted. These organ and tumor samples were weighed and digested by aqua regia solution for a week. After complete tissue digestion, each sample was diluted by water to 4 mL. Then, the Fe content in these samples was measured by ICP-OES for 3 times and the data were expressed as mean ± SD (n = 3).

Statistical analysis
One-way ANOVA statistical analysis was used to analyze the significance of the experimental data. A p value of 0.05 was selected as the level of significance, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.